The DNA in the nucleus is complexed with proteins to form chromatin and chromatin domains of variable accessibility reside in specific regions of the nucleus. This packaging and organization is important for normal chromosomal processes like transcription and disruption of this organization is known to lead to developmental defects and disease states. The overall objective of this proposal is to reach a mechanistic understanding of the structure and organization of chromatin domains in eukaryotic chromosomes. DNA elements are necessary for the formation of chromatin domains and these elements also function to organize the domains within the three-dimensional context of the nucleus. Understanding long-range interactions between DNA elements and their role in the dynamics of chromatin domain is one focus of this proposal. The final goal is to understand how chromatin bound factors and DNA elements influence chromatin structure thereby affecting processes such as transcription. Adjacent chromatin domains in the nucleus often have antagonistic structural or functional properties and DNA insulator elements separate these domains. Promoters of specific genes often function as insulators in yeast and Drosophila and we have determined the broad outlines of the mechanisms by which these insulators function in yeast. In this grant we propose to investigate the changes that occur in chromatin domains as cells divide and to determine if similar mechanisms operate to separate chromatin domains in human cells. The research will involve use of molecular genetics and genomics, manipulation of chromatin using techniques like quantitative chromatin immunoprecipitations, and chromosome conformation capture and biochemical analysis. Understanding how the nucleus is organized should help us better manipulate processes for benefit in research. The results from this study will be useful in understanding the molecular basis of disease states that arise from mis-regulation of chromatin domains. The results will also enable researchers to develop new strategies for treating disease states by developing new molecular tools for gene therapy.